Lysyl oxidase is an extracellular copper enzyme that initiates the crosslinking of collagens and elastin by catalyzing oxidative deamination of the xcex5-amino group in certain lysine and hydroxylysine residues of collagens and lysine residues of elastin (Kaman in Biology of Extracellular Matrix, ed. Mecham (1986) Academic Press pp. 321-389). Lysyl oxidase has been shown to be important in a variety of cellular and physiologic processes including biogenesis of connective tissue matrices and bone resorption. A deficiency in lysyl oxidase activity is found in two X-linked, recessively inherited connective tissue disorders, the type IX variant of the Ehlers-Danlos syndrome and the Menkes syndrome, and in the X-linked, recessively inherited mottled series of allelic mutant mice (all characterized by abnormalities in copper metabolism). (Byers et al. (1980) New Engl. J. Med. 303:61-65; Royce et al. (1980) Biochemistry J. 192:579-586; Kuivaniemi et al. (1982) J. Clin. Invest. 69:730-733; Kuivaniemi et al. (1985) Amer. J. Human. Genet. 37:798-808; Peltonen et al. (1983) Biochemistry 22:6156-6163; Rowe et al. (1977) J. Biol. Chem. 252:939-942; Starcher et al. (1977) Biochem. Biophys. Res. Commun. 78:706-712; Danks in The Metabolic Basis oflnherited Diseasexe2x80x9d, eds. Stanbury et al. (1983), McGraw-Hill pp. 1251-1268). Increased lysyl oxidase activity has been associated with fibrotic disorders such as atherosclerosis, hypertension, and liver and pulmonary fibrosis. (Kagan, supra).
More recently there have been identified proteins having structural and/or functional similarities to lysyl oxidase. For example, a lysyl oxidase-like protein, referred to herein as xe2x80x9cLOLxe2x80x9d, was identified from a human skin fibroblast cDNA library that contains extensive homology to several coding domains within the human lysyl oxidase mRNA which is believed to be involved in collagen maturation. (Kenyon et al. (1993) J. Biol. Chem. 268:18435-18437 and Kim et al. (1995) J. Biol. Chem. 270:7176-7182). Likewise, a protein referred to herein as lysyl-oxidase related protein (xe2x80x9cLorxe2x80x9d) has been identified which inhibits many of the structural features of lysyl oxidase and is overexpressed in senescent fibroblasts and is believed to play a role in age-associated changes in extracellular proteins. (Saito et al. (1997) J. Biol. Chem. 272:8157-8160). Lor contains four domains referred to herein as scavenger receptor cysteine-rich domains (xe2x80x9cSRCR domainsxe2x80x9d) which are believed to be involved in binding to other cell surface proteins or extracellular molecules. The SRCR domain joins a long list of other widely distributed cysteine-containing domains found in extracellular portions of membrane proteins and in secreted proteins (Doolittle (1985) Trends Biochem. Sci. 10:233-237; Krieger in Molecular Structures of Receptors, eds. Rossow et al. (1986) Horwood, Chichester, U.K. pp. 210-231). Examples include the EGF-like domain, immunoglobulin superfamily domains, the LDL receptor/complement. C9 domain, clotting factor Kringle domains, and fibronectin domains. These disulfide cross-linked domains appear to provide stable core structures that (i) are able to withstand the rigors of the extracellular environment; (ii) are well suited for a variety of biochemical tasks, often involving binding; and (iii) are readily juxtaposed to other types of domains to permit the construction of complex mosaic proteins. (Doolittle supra; Sudhof et al. (1985) Science 228:815-822). Lastly, a mouse cDNA encoding a putative protein having sequence homology to lysyl oxidase has recently been identified having the Accession No. AF053368, referred to herein as xe2x80x9cLor-2xe2x80x9d.
A greater understanding of the role which lysyl oxidase-like as well as SCRC domain containing proteins play in various disorders would lead to the determination of highly specific drug targets which would work to treat these disorders, e.g., cardiovascular disorders, a disorder arising from altered lysyl oxidase-like activity or a disorder arising from improperly regulated SRCR-domain containing protein activity giving rise to improperly regulated cellular processes.
The present invention is based, at least in part, on the discovery of novel nucleic acid molecules and proteins encoded by such nucleic acid molecules, referred herein as Myocardium Secreted Protein-18 (xe2x80x9cMSP-18xe2x80x9d) molecules. The MSP-18 nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes in the cardiovascular system, e.g., cardiac cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding MSP-18 proteins or portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of MSP-18-encoding nucleic acids. In another embodiment, an isolated nucleic acid molecule of the present invention preferably encodes a MSP-18 protein which includes a signal sequence and/or is secreted. In yet another embodiment, an isolated nucleic acid molecule of the present invention preferably encodes a MSP-18 protein which lacks a signal sequence and/or is intracellular.
In one embodiment, a MSP-18 nucleic acid molecule of the invention is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% 97%, 98%, 99%, or more homologous to a nucleic acid sequence (e.g., to the entire length of the nucleotide sequence) having the nucleotide sequence shown in SEQ ID NO:1 or a complement thereof.
In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:1, or a complement thereof. In another embodiment, the nucleic acid molecule includes nucleotides 143-2401 shown in SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule comprises a fragment of at least 50 contiguous nucleotides of the nucleotide sequence shown in SEQ ID NO:1, or a complement thereof.
In another embodiment, an MSP-18 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more homologous to the amino acid sequence shown in SEQ ID NO:2.
In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human MSP-18. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence shown in SEQ ID NO:2. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein at least 753 amino acids in length. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein at least 728 amino acids in length. In a further preferred embodiment, the nucleic acid molecule encodes a protein having an MSP-18 activity (as described herein).
Another embodiment of the invention features nucleic acid molecules, preferably MSP-18 nucleic acid molecules, which specifically detect MSP-18 nucleic acid molecules relative to nucleic acid molecules encoding non-MSP-18 proteins. For example, in one embodiment, such a nucleic acid molecule is at least 300, 400, 500, 600, 650, 700, 750, or 753 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or a complement thereof. In a particularly preferred embodiment, the nucleic acid molecule comprises a fragment of at least 50 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, or a complement thereof. In another preferred embodiment, the nucleic acid molecules are at least 25, 50, 75, 100, 150, 200, 250 or more nucleotides (e.g., contiguous) in length and hybridize under stringent conditions to SEQ ID NO:1. In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide having the amino acid sequence shown in SEQ ID NO:2, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:1 under stringent conditions.
Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a MSP-18 nucleic acid molecule, e.g., the coding strand of a MSP-18 nucleic acid molecule.
Another aspect of the invention provides a vector comprising a MSP-18 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. The invention also provides a method for producing a protein, preferably a MSP-18 protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.
Another aspect of this invention features isolated or recombinant MSP-18 proteins and polypeptides. In one embodiment, the isolated polypeptide includes one or more of the following: a signal sequence, a LOX domain and at least one SCRC domain. In another embodiment, the isolated polypeptide includes a signal sequence, a LOX domain and at least two, three, or four SCRC domains. In another embodiment, the isolated protein preferably includes a signal sequence, a LOX domain, at least one SCRC domain and has an amino acid sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to a protein having the amino acid sequence shown in SEQ ID NO:2. In yet another embodiment, the isolated protein, preferably a MSP-18 protein, includes a signal sequence, a LOX domain, at least one SCRC domain and is expressed and/or functions in cells of the cardiovascular system.
In yet another embodiment, an isolated protein, preferably a MSP-18 protein, has a signal sequence and/or is secreted. In another embodiment, the isolated protein, preferably a MSP-18 protein, includes a signal sequence, a LOX domain, at least one SCRC domain and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1.
In another embodiment, the isolated protein, preferably a MSP-18 protein, has an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to a polypeptide having the amino acid sequence shown in SEQ ID NO:2 (e.g., the entire amino acid sequence shown in SEQ ID NO:2). In another embodiment, the invention features fragments of the proteins having the amino acid sequence shown in SEQ ID NO:2, wherein the fragment comprises at least about 25, 50, 75, 100, 150, 200, 250 or more amino acids (e.g., contiguous amino acids) of the amino acid sequence shown in SEQ ID NO:2. In another embodiment, the protein, preferably a MSP-18 protein, has the amino acid sequence shown in SEQ ID NO:2.
Another embodiment of the invention features an isolated protein, preferably a MSP-18 protein, which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleic acid having the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1, or a complement thereof. This invention further features an isolated protein, preferably a MSP-18 protein, which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, or a complement thereof
The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-MSP-18 polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably MSP-18 proteins. In addition, the MSP-18 proteins or portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
In another aspect, the present invention provides a method for detecting the presence of a MSP-18 nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a MSP-18 nucleic acid molecule, protein or polypeptide such that the presence of a MSP-18 nucleic acid molecule, protein or polypeptide is detected in the biological sample.
In another aspect, the present invention provides a method for detecting the presence of MSP-18 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of MSP-18 activity such that the presence of MSP-18 activity is detected in the biological sample.
In another aspect, the invention provides a method for modulating MSP-18 activity comprising contacting a cell capable of expressing MSP-18 with an agent that modulates MSP-18 activity such that MSP-18 activity in the cell is modulated. In one embodiment, the agent inhibits MSP-18 activity. In another embodiment, the agent stimulates MSP-18 activity. In one embodiment, the agent is an antibody that specifically binds to a MSP-18 protein. In another embodiment, the agent modulates expression of MSP-18 by modulating transcription of a MSP-18 gene or translation of a MSP-18 mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a MSP-18 mRNA or a MSP-18gene.
In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant MSP-18 protein or nucleic acid expression or activity by administering an agent which is a MSP-18 modulator to the subject. In one embodiment, the MSP-18 modulator is a MSP-18 protein. In another embodiment the MSP-18 modulator is a MSP-18 nucleic acid molecule. In yet another embodiment, the MSP-18 modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant MSP-18 protein or nucleic acid expression is a cardiovascular disorder, e.g., congestive heart failure, ischemia, cardiac hypertrophy, ischemic-reperfusion injury or a disorder arising from improperly regulated MSP-18 protein action on target molecules/cells giving rise to improperly regulated cellular processes.
The present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a MSP-18 protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a MSP-18 protein, wherein a wild-type form of the gene encodes a protein with a MSP-18 activity.
In another aspect the invention provides a method for identifying a compound that binds to or modulates the activity of a MSP-18 protein, by providing an indicator composition comprising a MSP-18 protein having MSP-18 activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on MSP-18 activity in the indicator composition to identify a compound that modulates the activity of a MSP-18 protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.